Zataria multiflora Boiss. Essential Oil Induce Apoptosis in Two Human Colon Cancer Cell Lines (HCT116 & SW48)

Background: Zataria multiflora Boiss. is known by the common Persian name “Avishan-e-Shirazi”, is one of the best-known medicinal herbs belonging to the Labiatae (Lamiaceae) family. The aim of this study was to evaluate the anticancer effects and the underlying mechanisms of how Z. multiflora Boiss., essential oil induced apoptosis in the human colorectal tumor cell lines (HCT116 & SW48). Methods: This study was conducted in National Institute of Genetic Engineering and Biotechnology (NIGEB) (Tehran, Iran) from 2017 to 2019. The cytotoxicity of this essential oil was assessed by 3- (4,5-di-methylthiazol-2-yl)- 2,5-diphenyltetra-zolium bromide (MTT) assay, trypan blue exclusion, and colony formation assays. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Then gene expression was analyzed by Quantitative Real-Time RT-PCR. Results: Z. multiflora Boiss., essential oil time- and dose-dependently inhibits cell proliferation and also induced apoptosis in both cell lines via UCP2-related mitochondrial pathway by the induction of intracellular ROS. Conclusion: Z. multiflora Boiss., essential oil could be a good candidate for use as an inhibitor of the growth of colorectal tumor cells.


Introduction
Colorectal cancer (CRC) is the fourth leading cause of cancer death in women, behind breast, lung, and cervical cancer, respectively and second leading cause of cancer death in men, behind only lung cancer, in the world (1). There are different approaches to treat colorectal cancer, based on the type and stage of colorectal cancer, including surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy etc. (1). Natural compounds derived from plants have been regarded as potential chemopreventive and chemotherapeutic compounds in the last decade (2). Due to they are relatively less toxic to healthy cells, and they are cost-effective and better accepted by patients, they are now playing an attractive role for the development of tumor treatment and prevention strategies (3,4). Zataria multiflora's English name is Wild marjoram and its botanical name is Zataria multiflora Boiss and is known by the common Persian name "Avishan-e-Shirazi". Z. multiflora Boiss., is one of the bestknown medicinal herbs belonging to the Labiatae (Lamiaceae) family widely distributed in Iran, Pakistan and Afghanistan (5). This plant is not only a popular food flavor ingredients but is also well known for its diverse beneficial effects, including pharmaceutical, antimicrobial, anti-nociceptive, antioxidant and anti-inflammatory properties (6). The biological properties of Z. multiflora have been attributed to the essential oil that has phenolic compounds such as carvacrol and thymol. Z. multiflora essential oil is extracted from the flowered branches of the plant (5,6). This essential oil is a growth inhibitor that currently bears clinical assessment for several malignancies such as cervical cancer, breast cancer (7,8). However, the effect of this essential oil still needs to be elucidated in colorectal cancers. UCP2 belongs to a family of anion carrier proteins located in the inner mitochondrial membrane (9). UCP2 is over-expressed in various types of cancer. UCP2 negatively regulates the production of reactive oxygen species (ROS) in the mitochondrion (10,11). The BCL-2 family members can be divided into three groups: antiapoptotic proteins (such as BCL-2 (B-cell lymphoma 2), BCL-XL (B-cell lymphoma-extralarge)), pro-apoptotic effectors (such as Bax (BCL2 associated X protein ), Bak (BCL2 antagonist/killer)), and pro-apoptotic activators (such as Bad (Bcl2-associated agonist of cell death), Bik (BCL2 interacting killer)) (11). Therefore, we assessed the expression of Ucp2, Bcl2, Bax, Bak, bik, Cytochrome c genes. The present study aimed to investigate the cytotoxic effects and also the underlying mechanisms of how Z. multiflora Boiss., essential oil induced apoptosis in human colorectal cancer cell lines (HCT116 & SW48).

Treatment of cells with Z. multiflora Boiss. essential oil
Z. multiflora Boiss., essential oil was purchased from Barij Essence Pharmaceutical Company, Kashan, Iran. Next, serial dilutions of this essential oil were prepared in culture medium, DMEM and RPMI 1640, to get the desired concentrations ranged from 2.5 to 250 ppm.

Microculture Tetrazolium (MTT) Assay
To determine cell viability 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay was used. HCT116 and SW48 cell lines (8000 cells/well) were seeded in 96-well plates (SPL Life Sciences, Pocheon, Korea) and incubated in a CO2 incubator at 37 °C. After 24 h the original medium was removed and 100 μl fresh medium was added, then cells were treated with Z. multiflora Boiss. essential oil (with concentrations of 0, 2.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250 ppm) and incubated for 24, 48 and 72 h, after this time the medium was replaced with ready-To-Use RPMI 1640. Then 10 μl of the 12mM MTT solution (BIO-IDEA, IRAN) (Cat No. BI1017) was added to each well and the plates were incubated for 4 h. After that, medium was removed from the wells, and then 50 μl of Dimethyl sulfoxide (DMSO) solution was added to each well, to solve precipitated formazan. Then plates were incubated for 10 min. Finally, the optical densitometry was measured by ELISA reader at a wavelength of 570 nm. The percentage of cell viability was calculated as (%)=(optical density of treated cells/optical density of untreated cells) × 100. The survival curves of HCT116 and SW48 cell lines were constructed based on different concentrations of extract after the specified period. The effects of essential oil were determined by IC50 values (the concentration of essential oil that reducing the absorbance of treated cells by 50% with respect to untreated cells) (12,13).

Trypan Blue Exclusion Test
To discriminate between viable and non-viable cells, trypan blue exclusion test was used. Approximately 1 × 10 5 of HCT116 & SW48 cells were seeded per well in 6-well plates and incubated. After 24 h, the cells were treated with Z. multiflora Boiss., essential oil (with concentrations of 0, 20, 40, 50, 100, 250 ppm) and incubated. Overall, 48 h after treatment, Cells were harvested by digestion with 0.25% trypsin-EDTA solution (1X) (BIO-IDEA, IRAN) (Cat No. BI1002). Then, harvested cells were resuspended in phosphatebuffered saline (PBS) (BIO-IDEA, IRAN) (Cat No. BI-1038-500), and then 10 µl of cell suspension were mixed with 10 µl of 0.4% trypan blue dye (BIO-IDEA, IRAN) (Cat No. BI1014), then they were mixed and incubated at room temperature for 5 min. After that, viable cells that had clear cytoplasm (colorless) and non-viable cells that had blue cytoplasm were counted by using a Neubauer hemocytometer. Finally, percentage of viable cells was calculated as follows: Cell Viability (%)=number of viable cells ÷ number of total cells × 100. Cell counts were expressed as mean ± standard deviation (SD).

Colony Formation Assay (CFA)
In vitro cell survival assay based on the ability of a single cell to grow into a colony is named Clonogenic Assay or Colony Formation Assay. To evaluate the effects of Z. multiflora Boiss., essential oil on colony forming potential of HCT116 and SW48 cell lines, Colony Forming Assay was performed. For this, 1000 cells per well were seeded on a 6-well plate and incubated. Overall, 24 h after incubation, media were removed and cells were rinsed with PBS, then cells were treated with Z. multiflora Boiss., essential oil (with concentrations of 0, 20, 40, 50, 100, 250 ppm) and incubated for 48 h. Then, the media were replaced by fresh media without drug and the cells were incubated for 18 d until cells in control plates have formed colonies. After 18 d of incubation, cultured cells were washed with PBS, thereafter, the plates were fixed with methanol: acetic acid (3:1) for 10 min, and stained with 0.5% crystal violet and left at room temperature for 30 min to dry (14). The colonies were counted using an inverted microscope (Zeiss, Axiovert 405M; Germany). Colonies, defined as 50 or more cells, were counted and plotted as a percentage of the control (untreated) colonies (15).

Evaluation of cellular morphology
The effects of the Z. multiflora Boiss., essential oil on the morphology of HCT116 and SW48 colorectal cell lines were observed using phasecontrast microscopy and were photographed prior to and after treatment with IC50 values of this essential oil, to document possible changes in morphology.

Acridine Orange (AO) and Ethidium Bromide (EB) Double Staining
EB/AO stain determines the membrane integrity of a cell-based on the uptake or exclusion of a dye from the cell (16). Ethidium bromide is a dye that stains only cells that have lost membrane integrity and makes cells red. Acridine orange is a membrane-permeable dye that will stain both live and dead cells and makes cells green (17). The effects of Z. multiflora Boiss., essential oil on HCT116 and SW48 cell lines were determined morphologically by fluorescent microscopy, after labeling with acridine orange (MERK) (Cat No. 113000) and ethidium bromide. For this purpose, HCT116 and SW48 cells were seeded in six-well plates and incubated. After 24 h, cells were treated with 0, 50, 100, 200 ppm of Z. multiflora Boiss., essential oil and incubated. 48 h after treatment, cell suspension was collected by centrifugation and washed with PBS and then resuspended in 10 μl of dye mixture containing 10.0 μL of Acridine orange (10.0 μg/mL) and 10.0 μL of Ethidium bromide (10.0 μg/mL) and mixed gently. Then, 10.0 μL of mixed suspension was placed on a microscope slide and covered with coverslips and the morphological changes examined under fluorescence microscope.

Measuring Apoptosis Using Flow Cytometry
Apoptosis was measured by Annexin V-FITC and propidium iodide (Annexin-V-FITC Apoptosis Detection Kit, Cat. No.: A2214-Sigma). HCT116 and SW48 cells were seeded at approximately 1 × 10 5 cells per well in 6-well plates and allowed to adhere in 24 h at 37 °C in 5% CO2 incubator. Then old medium was replaced with fresh medium plus 10% FBS and supplemented with 50 ppm and 42 ppm of Z. multiflora Boiss. essential oil respectively for HCT116 and SW48 cells and incubated. 48 h after treatment, Cells were harvested and washed with PBS and resuspended in 2000 L of 1X Binding Buffer then gently mixed. After that 500 L of cell suspension was transferred to four 5 ml culture tube. The first tube is cells without stain. To second and third tube, 5 L Annexin V-fluorescein isothiocyanate (Annexin V) was added. All the tubes incubated at room temperature for 15 min in the dark. Then to third and fourth tubes 3 L of Propidium Iodide (PI) was added. The samples were analyzed on a BD FACS Calibur flow cytometer (Becton, Dickinson and Company, Palo Alto, CA, USA). Data analysis was performed with FlowJo software (Becton, Dickinson, and Company).

Determination of intracellular reactive oxygen species (ROS) Production
HCT116 and SW48 cells were seeded onto 6-well plates (at approximately 1 × 10 5 cells/well) and incubated overnight. Then, cells were treated respectively with 50 ppm and 42 ppm of Z. multiflora Boiss. essential oil and incubated. After 48 h, Cells were harvested and washed twice with PBS. 100 μM of 1X 2′,7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO) was added to the cells and incubated for 45 min. Samples immediately were analyzed on a BD FACS Calibur flow cytometer (extinction wavelength: 485 nm and emission wavelength: 530 nm).

Gene expression analysis by real-time quantitative PCR
3×10 3 of HCT116 and SW48 cells were seeded in T25 flask and incubated at 37 o C in 5% CO2. Next day, cells were stimulated with IC50 concentrations of Z. multiflora Boiss. essential oil and incubated for 48 h. Then total RNA was isolated using RNX-Plus solution (Cat. No.: RN7713C, Cinnagen, Iran) according to manufacturer's instructions. The quantity and quality of extracted RNA were respectively assessed by Nanodrop and by running 3µl of RNA in 1% agarose gel. Then, 500 ng of RNA from each sample was reversely transcripted by using Easy cDNA Synthesis Kit (Pars tous, Iran). Real-time PCR was performed with a Rotor-Gene Q system (Cat. No.: 9001560, Qiagen, Germany) using YTA SYBER Green qPCR Master Mix 2X (Cat. No.: YT2551, Yekta Tajhiz Azma, Iran). In a total volume of 20 in a capillary tube, 300 ng of cDNA samples, 10 μl SYBR Green Master Mix, 0.5 μl of forward and reverse primers (10 pmol), and up to 20 of nuclease-free water (Qiagen, Hilden, Germany) were added. To validate single PCR product of each primer, melting curves were analyzed. The oligonucleotide sequences and relevant product size are listed in Table 1, and GAPDH gene was selected as an internal reference. The primer sets were purchased from TAG Copenhagen (Copenhagen, Denmark). Relative expression was calculated using the Livac or 2 -ΔΔCt .

Statistical Analysis
For statistical analysis SPSS 22 Software (Chicago, IL, USA) was used. The data are expressed as mean ± standard deviation (SD). All the experiments in this work were performed in duplicate or triplicate. First, for the assessment of normality, we used Kolmogorov-smirnov(K-S) test. Our data are non-normal. Because they are obtained from a non-normal distribution, Kruskal-Wallis test and by Mann-Whitney test, that both are nonparametric test, were used. The statistical significance of differences throughout this study was calculated by Kruskal-Wallis test and by and Pvalue ≤0.05 was considered statistically significant.

Results
The results of MTT assay showed that the treatment of cells moderately reduced metabolic activ-ity of both cell lines in dose and time-dependent manner (Fig. 1).     (Fig. 1). The results of clonogenic assay showed that treatment with Z. Multiflora Boiss. essential oil significantly decreased the capacity of these tumor cells to establish colonies to a highly significant degree in a concentration dependent manner (Fig. 2). Moreover, the results of colony formation assay confirmed data of MTT and Trypan blue exclusion assays. Direct observation under a light microscope was used to assess the morphological changes of HCT116 and SW48 cell cultures after treatment with Z. Multiflora Boiss. essential (Fig. 3). In present study, the apoptosis, necrosis and viability of colorectal cancer cell lines treated with this essential oil were evaluated under a fluorescence microscope using acridine orange and ethidium bromide stains (Fig. 3). Cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation were noticed in the HCT116 and SW48 cells after 48 h of treatment, indicating that Z. multiflora Boiss. essential oil induced apoptosis in these cells. In this study, Z. multiflora Boiss. essential oil was found to induce apoptosis in human colorectal cancer cell lines according to the results of annexin V/PI staining assay. In the cytograms, cells were untreated (up) and cells were treated with IC50 of Z. multiflora Boiss. essential oil (down) for 48 h; then, apoptosis was measured by flow cytometric analysis of cells labeled with Annexin V(x-axis) and propidiumiodide  (Fig. 4). Treatment of HCT116 and SW48 cells with Z. multiflora Boiss. essential oil for 48h, increased cellular ROS levels (Fig. 5).
Totally, 48 h after incubation, average fluorescences intensity in untreated HCT116 cells and in HCT116 cells that treated with 50 ppm of Z. multiflora Boiss. essential oil respectively were 525 and 2224, and in untreated SW48 cells and in SW48 cells that treated with 42 ppm of Z. multiflora Boiss. essential oil respectively was 148 and 159. Expression of Ucp2 in HCT116 and SW48 relative to normal human fibroblast cell lines was significantly increased, and expression of Ucp2 after treatment with Z. multiflora Boiss. essential oil was significantly decreased. After treatment, expression of anti-apoptotic gene Bcl2 was significantly decreased and the expression of proapoptotic effectors genes Bax, Bak and proapoptotic activators gene Bik was significantly increased in HCT116 and SW48 cell lines.

Discussion
This investigation showed that Z. multiflora Boiss. essential oil had anticancer activity against both HCT116 and SW48 cells and induced apoptosis via the UCP2-mediated mitochondrial pathway. After treatment of HCT116 and SW48 cells with Z. multiflora Boiss. essential oil, the expression of Ucp2 was downregulated. Following downregulation of Ucp2, the expression of Bcl2 was downregulated and the expression of Bax, Bak, Bik, Cytochrome c was upregulated in human colorectal HCT116 and SW48. Among plant derived compounds, essential oils (EOs) are volatile compounds produced by plants that present different biological activities including antitumor effect. In the last two decades, the anticancer potential of Essential oils and their components were explored in vitro and in vivo models (2). The essential oil of Z. Multiflora Boiss., was acquired by hydrodistillation and de-composed it by Gas Chromatography and Gas Chromatography -Mass Spectrometry. It was discovered 48 ingredients in this essential oil as the main ingredients were Carvacrol (More than half of it), Linalool, p-Cymene, Thymol (18). Z. Multiflora from Shiraz Province of Iran was discovered the main ingredients of Z. multiflora Boiss. essential oil was Carvacrol (71.1%) (5). Moreover, carvacrol have strong antitumor activity. For example, the anti-tumor effects of carvacrol was demonstrated on human metastatic breast cancer cells, MDA-MB 231 (2). The mRNA expression level of UCP2 gene was assessed in colon cancer and they found that UCP2 gene was upregulated in colon cancer tissue samples than in its adjacent tissue samples. They also revealed that the high UCP2 expression level was significantly correlated to colon cancer metastasis (19). Cytotoxic effects of Hydro-alcoholic extract of Z. multiflora was reported on HT-29 and SW-48 colon cancer cell lines using MTT assay (20).